Model for fluid and mass transport in a recirculating microfluidic system

ABSTRACT

Disclosed herein are microfluidic systems with recirculation of fluid and computer-implemented methods of calculating conditions within the microfluidic systems. The microfluidic systems include a computing device and a microfluidic device having first and second reservoirs, at least one chamber, and a fluid path connecting the first reservoir, the chamber, and the second reservoir. The methods for calculating conditions include receiving a first reservoir fluid volume, a second reservoir fluid volume, a first concentration, and a second concentration. The methods further include receiving a time-dependent imposed pressure difference between the first reservoir and the second reservoir, then determining a hydraulic pressure difference and an effective pressure difference. The effective pressure difference is used to account for reactions occurring within the microfluidic device and to determine the value of the condition within the microfluidic device. Methods of performing an experiment using a microfluidic device with recirculation are also disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/381,660, filed on Apr. 11, 2019, which claims benefit of U.S.Provisional Application No. 62/655,909, which was filed Apr. 11, 2018,the contents of which are hereby incorporated in their entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant no.R01NS050452 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The major research uses of animals are both in assessing potentialtoxicity of chemicals and in drug testing. Animal tests often are longin duration, expensive, and raise ethical issues. Further, animal testsare not always predictive of human response. In terms of human responseto environmental toxicants, it is not ethically possible to conductdirect tests on humans, and extrapolation of animal results to humanresponse is problematic. Over-regulation results in unnecessary expense;under-regulation endangers human health and the environment, so bettertesting systems are necessary.

In vitro tests can supplement and may reduce dependency on animal tests.However, in vitro tests fail to capture many important aspects of humanand mammalian response to chemicals. Most in vitro tests are based onthe use of multi-well plates where isolated cells or tissues are placedin medium spiked with a bolus dose of the test chemical. Such systemsmiss key aspects of physiological response. For example, the dosedynamics in the body differ considerably from static systems astime-dependent changes in chemical concentration occur in the body at atissue site due to the processes controlling absorption, distribution,metabolism and excretion of a compound. Further, static well systemstypically use a single cell or tissue type; in the body, metabolites areexchanged between different tissue/organ compartments. Even if multipletissue types were represented in a single well, the ratio of one tissueto another and the nature of the circuits connecting them can alter thetime-dependent concentration of the metabolites. In addition to thesefactors, single cells in a well, in most cases, do not representfunctional tissues or subsystems of the body, nor experience themechanical forces the cells in the body do, particularly thoseassociated with fluid flow, and these mechanical forces are known toalter gene expression and metabolism of many chemicals. Microfluidicdevices can be used to more accurately simulate true physiologicalsystems, enabling continuous flow of media past a cell culture and moreaccurately simulating physiological dose dynamics. These devices can bebuilt to incorporate compartments with different cell types orengineered organs.

SUMMARY

While microfluidic devices are able to more accurately simulate truephysiological systems than static well cell cultures, there is adifficulty in assessing conditions within a microfluidic device at amicroscopic level, particularly when the microfluidic systemrecirculates fluid. For example, reactions within the microfluidicdevice can be highly localized. A metabolite created by a cell culturein one chamber may become bound or react with an enzyme or cell in aseparate chamber, leading to disparities in local concentrations of themetabolite across the microfluidic device. Flow rates can also changebased on the structure of a particular chamber, leading to a poorlydefined shear stress acting upon cell cultures or other surfacecomponents within the device. The lack of understanding of theseconditions leads to expensive trial-and-error based experimentation.Thus, in the technical environment of recirculating biomedicalmicrofluidics, there is a need for systems that can pinpoint highlylocalized concentrations and flow rates using fluid and mass transportmodeling.

For body-on-a-chip biomedical microfluidic systems, different organs arerepresented by different units of the microfluidic. Recirculation withina biomedical microfluidic is desirable for several reasons. First,recirculation of the media enables interaction between the various unitsof a biomedical microfluidic system in a bidirectional manner. Bycontrast, systems with no recirculation enable interaction only in aunidirectional manner (that is, an upstream unit affects a downstreamunit, but the downstream unit does not affect the upstream unit).Additionally, recirculation enables the units of a biomedicalmicrofluidic to interact with each other over a longer time frame, suchthat slow interactions (e.g., slow chemical reactions) can build up overtime and affect the other units. For body-on-a-chip systems,bidirectional and long term interactions between units are important toaccurately simulate the body because recirculation of blood enablesinteractions among organs in complex and time-dependent manners.

Disclosed herein are microfluidic systems with recirculation of fluidand computer-implemented methods of calculating conditions within themicrofluidic systems. The microfluidic systems include a microfluidicdevice and a computing device. The microfluidic device includes a fluid,first and second reservoirs, at least one chamber in fluid communicationwith the first reservoir and the second reservoir, and a fluid pathconnecting the first reservoir, the chamber, and the second reservoir.The computing device includes a processor and a memory that is operablyconnected to the processor. The memory includes computer-executableinstructions that cause the processor to carry out the methods forcalculating the conditions within the microfluidic device.

Methods for calculating conditions within the microfluidic device havingrecirculation are also disclosed herein. Conditions can include, but arenot limited to, the concentration of a component at a specific spatiallocation, a concentration profile over a specified spatial region, aneffective flow rate of the fluid between the first and secondreservoirs, mass transport of chemical species or suspended components(e.g., particles) from one chamber to another, shear stresses onunits/objects/tissues in the microfluidic device, shear rates, shearstresses at the material interfaces, flow rates among the microfluidicchambers, and the average molecular velocity of the component. Themethods for calculating conditions include receiving, at the processor,a first reservoir fluid volume and a second reservoir fluid volume. Themethods further include receiving a first concentration, which is theconcentration of a component in a first reservoir, and a secondconcentration, which is the concentration of the same component in asecond reservoir. The methods further include receiving a time-dependentimposed pressure difference between the first reservoir and the secondreservoir, then determining a hydraulic pressure difference and aneffective pressure difference, which is based on the imposed andhydraulic pressure differences. The effective pressure difference isused to account for reactions occurring within the microfluidic deviceand to determine the value of the condition within the microfluidicdevice.

Methods of performing an experiment using a microfluidic device withrecirculation are also disclosed herein. The methods include setting adesired value for a condition within the microfluidic device, performinga calculation that accounts for an effective pressure difference betweenthe first reservoir and the second reservoir and reactions occurringwithin the microfluidic device, and determining a set of initialconditions for achieving the desired value.

It should be understood that the above-described subject matter may alsobe implemented as a computer-controlled apparatus, a computer process, acomputing system, or an article of manufacture, such as acomputer-readable storage medium.

Other systems, methods, features and/or advantages will be or may becomeapparent to one with skill in the art upon examination of the followingdrawings and detailed description. It is intended that all suchadditional systems, methods, features and/or advantages be includedwithin this description and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The components in the drawings are not necessarily to scale relative toeach other.

FIG. 1 shows an example implementation of a microfluidic systemincluding a microfluidic device with recirculation, a tilting platform,a pneumatic system, and a computing device.

FIG. 2 shows an example implementation of a microfluidic device.

FIG. 3 shows another example implementation of a microfluidic device.

FIG. 4 shows a schematic of a computing device.

FIG. 5 shows a flow chart illustrating a method of calculating acondition within a microfluidic device having recirculation according toan implementation described herein.

FIG. 6 shows a flow chart illustrating a method of performing anexperiment using a microfluidic device having recirculation according toan implementation described herein.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure.As used in the specification, and in the appended claims, the singularforms “a,” “an,” “the” include plural referents unless the contextclearly dictates otherwise. The term “comprising” and variations thereofas used herein is used synonymously with the term “including” andvariations thereof and are open, non-limiting terms. The terms“optional” or “optionally” used herein mean that the subsequentlydescribed feature, event or circumstance may or may not occur, and thatthe description includes instances where said feature, event orcircumstance occurs and instances where it does not. Ranges may beexpressed herein as from “about” one particular value, and/or to “about”another particular value. When such a range is expressed, an aspectincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another aspect. It will be further understood that the endpointsof each of the ranges are significant both in relation to the otherendpoint, and independently of the other endpoint. As used herein,“operably connected” means that the particular elements are connected insuch a way that they cooperate to achieve their intended function orfunctions. The “connection” may be direct, indirect, physical, orremote.

Example Microfluidic Devices

Disclosed herein are microfluidic systems with recirculation of fluidand computer-implemented methods of calculating conditions within themicrofluidic systems. As shown in FIG. 1 , microfluidic system 100include a microfluidic device 200 and a computing device 400. Exemplarymicrofluidic devices 200 are shown in FIGS. 2 and 3 . The microfluidicdevices 200 include first and second reservoirs 202, 204, one or morechambers 206 in fluid communication with the first reservoir 202 and thesecond reservoir 204, a fluid path 208 connecting the first reservoir202, the chamber(s) 206, and the second reservoir 204, and a fluid thatflows along the fluid path 208 between the first reservoir 202, thechamber(s) 206, and the second reservoir 204 (fluid not shown). Ports218 may be used to deliver any fluid (liquid or gas) to the device 200.Ports 218 are shown as being fluidically connected to reservoirs 202,204. However, ports 218 can be connected at any position on themicrofluidic device 200. In some implementations, ports 218 can be usedto connect microfluidic devices to each other in series, such that therecirculating fluid is shared across devices.

In some implementations, the microfluidic system 100 can include atilting platform 212, as shown in FIG. 1 . The microfluidic device 200is placed upon the tilting platform 212 to create gravitational pressuredifferences that result in flow of the fluid within the microfluidicdevice 200. Alternatively, or in addition, a pneumatic pressure system214 can impose hydraulic pressure on the fluid within the microfluidicdevice 200, causing fluid flow. In one example, the pneumatic pressuresystem 214 can include tubing 216 to connect to a port 218 on themicrofluidic device 200. In some implementations, one or morecontrollers 220 can be included to receive instructions from computingdevice 400 regarding the imposed pressure difference and to regulate thepressure difference that is imposed upon the fluid, either by alteringthe tilting of platform 212 or by regulating the pneumatic pressuresystem 214.

The fluid of the microfluidic system 100 can include one or morecomponents at any desired concentration. In some implementations, thecomponents can be cells suspended within the fluid. The component couldalso be any large or small molecule, including polypeptides,polynucleotides, metabolites, cytokines, media reagents, enzymes, andantibodies. For example, in some implementations, the fluid is aserum-free medium, and the components include ingredients of theserum-free medium.

As shown in FIGS. 2 and 3 , the microfluidic device can include one ormore chambers 206 (e.g., chambers 206 a, 206 b in FIG. 2 or chamber 206in FIG. 3 ). The chambers can, for example, include one or morefunctional units 222 that can include cells or other biologicallyreactive surface components as described below. In some implementations,the functional units 222 can be considered to be analogous to a tissue,or they can play a role in a simulated organ or organ system. In someimplementations, a functional unit 222 can include a surface, substratecontainer or three-dimensional construct for cells (i.e., in which cellsare contained, grown, acted on and/or maintained). For example, themicrofluidic device 200 shown in FIG. 2 comprises a first functionalunit 222 a comprising a liver cell culture housed within athree-dimensional construct 224. Fluid, for example, a serum-free cellculture media, from the first reservoir 202 enters a first chamber 206 acontaining the three-dimensional construct 224 to deliver nutrients tothe liver cell culture housed therein. The fluid exits the first chamber206 a via fluid path 208 and enters a second chamber 206 b housing asecond functional unit 222 b that comprises a neuromuscular junction 226between a culture of motoneurons on a microelectrode array 228 and amuscle cell culture on a cantilever array 230. The first and secondchambers 206 a, 206 b are collectively referred to herein as chambers206. International Patent Application No. PCT/US17/47085 and U.S. PatentApplication Publication No. 2015/0219622 teach microfluidic systemscomprising a variety of example functional units, and are eachincorporated by reference in their entireties.

A surface component 232 can be attached to any two or three-dimensionalsurface within the microfluidic device. The surface component can reactwith components of the fluid, for example, cells, polypeptides,polynucleotides, metabolites, cytokines, media reagents, enzymes, andantibodies. In some implementations, the surface component 232 is partof a functional unit 222. In some implementations, such as the one shownin FIG. 2 , the surface component 232 is a cell or cell culture (such asthe liver cells 232 a within three-dimensional construct 224, themotoneurons 232 b on the microelectrode array 228, or the muscle cells232 c on the cantilever array 230). The surface component 232 can be,for example, immobilized antibodies (as shown in FIG. 3 ), immobilizedenzymes, or any other molecule, peptide, or protein that can react withcomponents of the fluid.

Example Computing Devices

A schematic of the computing device 400 is shown in FIG. 4 . Thecomputing device 400 includes a processor 406 and a memory 404 that isoperably connected to the processor 406. The memory includescomputer-executable instructions that cause the processor 406 to carryout the methods for calculating the conditions within the microfluidicdevice. It should be appreciated that the logical operations describedherein with respect to the various figures may be implemented (1) as asequence of computer implemented acts or program modules (i.e.,software) running on a computing device (e.g., the computing devicedescribed in FIG. 4 ), (2) as interconnected machine logic circuits orcircuit modules (i.e., hardware) within the computing device and/or (3)a combination of software and hardware of the computing device. Thus,the logical operations discussed herein are not limited to any specificcombination of hardware and software. The implementation is a matter ofchoice dependent on the performance and other requirements of thecomputing device. Accordingly, the logical operations described hereinare referred to variously as operations, structural devices, acts, ormodules. These operations, structural devices, acts and modules may beimplemented in software, in firmware, in special purpose digital logic,and any combination thereof. It should also be appreciated that more orfewer operations may be performed than shown in the figures anddescribed herein. These operations may also be performed in a differentorder than those described herein.

Referring to FIG. 4 , an example computing device 400 upon whichimplementations of the invention may be implemented is illustrated. Itshould be understood that the example computing device 400 is only oneexample of a suitable computing environment upon which implementationsof the invention may be implemented. Optionally, the computing device400 can be a well-known computing system including, but not limited to,personal computers, servers, handheld or laptop devices, multiprocessorsystems, microprocessor-based systems, network personal computers (PCs),minicomputers, mainframe computers, embedded systems, and/or distributedcomputing environments including a plurality of any of the above systemsor devices. Distributed computing environments enable remote computingdevices, which are connected to a communication network or other datatransmission medium, to perform various tasks. In the distributedcomputing environment, the program modules, applications, and other datamay be stored on local and/or remote computer storage media.

In its most basic configuration, computing device 400 typically includesat least one processor 406 and system memory 404. Depending on the exactconfiguration and type of computing device, system memory 404 may bevolatile (such as random access memory (RAM)), non-volatile (such asread-only memory (ROM), flash memory, etc.), or some combination of thetwo. This most basic configuration is illustrated in FIG. 4 by dashedline 402. The processor 406 may be a standard programmable processorthat performs arithmetic and logic operations necessary for operation ofthe computing device 400. The computing device 400 may also include abus or other communication mechanism for communicating information amongvarious components of the computing device 400.

Computing device 400 may have additional features/functionality. Forexample, computing device 400 may include additional storage such asremovable storage 408 and non-removable storage 410 including, but notlimited to, magnetic or optical disks or tapes. Computing device 400 mayalso contain network connection(s) 416 that allow the device tocommunicate with other devices. Computing device 400 may also have inputdevice(s) 414 such as a keyboard, mouse, touch screen, etc. Outputdevice(s) 412 such as a display, speakers, printer, etc. may also beincluded. The additional devices may be connected to the bus in order tofacilitate communication of data among the components of the computingdevice 400. All these devices are well known in the art and need not bediscussed at length here.

The processor 406 may be configured to execute program code encoded intangible, computer-readable media. Tangible, computer-readable mediarefers to any media that is capable of providing data that causes thecomputing device 400 (i.e., a machine) to operate in a particularfashion. Various computer-readable media may be utilized to provideinstructions to the processor 406 for execution. Example tangible,computer-readable media may include, but is not limited to, volatilemedia, non-volatile media, removable media and non-removable mediaimplemented in any method or technology for storage of information suchas computer readable instructions, data structures, program modules orother data. System memory 404, removable storage 408, and non-removablestorage 410 are all examples of tangible, computer storage media.Example tangible, computer-readable recording media include, but are notlimited to, an integrated circuit (e.g., field-programmable gate arrayor application-specific IC), a hard disk, an optical disk, amagneto-optical disk, a floppy disk, a magnetic tape, a holographicstorage medium, a solid-state device, RAM, ROM, electrically erasableprogram read-only memory (EEPROM), flash memory or other memorytechnology, CD-ROM, digital versatile disks (DVD) or other opticalstorage, magnetic cassettes, magnetic tape, magnetic disk storage orother magnetic storage devices.

In an example implementation, the processor 406 may execute program codestored in the system memory 404. For example, the bus may carry data tothe system memory 404, from which the processor 406 receives andexecutes instructions. The data received by the system memory 404 mayoptionally be stored on the removable storage 408 or the non-removablestorage 410 before or after execution by the processor 406.

It should be understood that the various techniques described herein maybe implemented in connection with hardware or software or, whereappropriate, with a combination thereof. Thus, the methods andapparatuses of the presently disclosed subject matter, or certainaspects or portions thereof, may take the form of program code (i.e.,instructions) embodied in tangible media, such as floppy diskettes,CD-ROMs, hard drives, or any other machine-readable storage mediumwherein, when the program code is loaded into and executed by a machine,such as a computing device, the machine becomes an apparatus forpracticing the presently disclosed subject matter. In the case ofprogram code execution on programmable computers, the computing devicegenerally includes a processor, a storage medium readable by theprocessor (including volatile and non-volatile memory and/or storageelements), at least one input device, and at least one output device.One or more programs may implement or utilize the processes described inconnection with the presently disclosed subject matter, e.g., throughthe use of an application programming interface (API), reusablecontrols, or the like. Such programs may be implemented in a high levelprocedural or object-oriented programming language to communicate with acomputer system. However, the program(s) can be implemented in assemblyor machine language, if desired. In any case, the language may be acompiled or interpreted language and it may be combined with hardwareimplementations.

Example Methods

A flow chart of an example method for calculating a condition within amicrofluidic device having recirculation is shown in FIG. 5 . As usedherein, the term “receive” can refer to the receipt of external orinternal inputs by the processor 406. For example, in someimplementations, the processor 406 receives an external input of thefirst reservoir fluid volume from a user such as a researcher who isplanning an experiment. It should be understood that the user canprovide such information using the input device 414. In someimplementations, the processor 406 receives an internal input of thefirst reservoir fluid volume as part of a larger, iterative method, forexample, when attempting to find the necessary initial condition (thefirst reservoir fluid volume) to obtain a desired value of a conditionwithin the microfluidic device (which will be described below).Conditions being calculated can include, but are not limited to, theconcentration of a component at a specific spatial location, aconcentration profile over a specified spatial region, an effective flowrate of the fluid between the first and second reservoirs, masstransport of chemical species or suspended components (e.g., particles)from one chamber to another, shear stresses on units/objects/tissues inthe microfluidic device, shear rates, shear stresses at the materialinterfaces, flow rates among the microfluidic chambers, and the averagemolecular velocity of the component. The methods for calculatingconditions include step 502 of receiving (at the processor 406) a firstreservoir fluid volume and step 504 of receiving a second reservoirfluid volume. The method further includes step 506 of receiving a firstconcentration, which is the concentration of a component in a firstreservoir. As described above, a component is any cell or molecule(large or small), including polypeptides, polynucleotides, metabolites,cytokines, enzymes, and antibodies. The method also includes step 508 ofreceiving a second concentration, which is the concentration of the samecomponent in the second reservoir. The methods further include step 510of receiving a time-dependent imposed pressure difference between thefirst reservoir and the second reservoir, step 512 of determining ahydraulic pressure difference, and step 514 of determining an effectivepressure difference. The hydraulic pressure difference is based on afirst volumetric height of fluid in the first reservoir and a secondvolumetric height of fluid in the second reservoir, and the effectivepressure difference is which is based on the imposed and hydraulicpressure differences. The effective pressure difference is used in step516 when accounting for reactions occurring within the microfluidicdevice that deplete or increase the first and second concentrations ofthe component. Finally, in step 518, the value of the condition withinthe microfluidic device is determined.

In some implementations of the methods, the imposed pressure differencechanges over at least one time step. The processor can receive theimposed pressure difference, the first reservoir fluid volume, thesecond reservoir fluid volume, the first concentration, and the secondconcentration before the time step (or before and after the time step)to determine the value of the condition. In some implementations, thevalue of the condition is iteratively determined before and after eachof a plurality of time steps. The change in the imposed pressuredifference can follow a regular waveform, such as a sawtooth,sinusoidal, or square waveform. Alternatively, the imposed pressuredifference can follow a non-regular pattern. For example, the timing ofthe tilting platform 212 shown in FIG. 1 can be set to cause a regularor non-regular change in the imposed pressure by changing thegravitational forces that affect the flow of the fluid within thedevice. Alternatively, or in addition, a pneumatic pressure system 214can be set to cause a regular or non-regular hydraulic pressure changewithin the microfluidic system. In some implementations, the computingdevice 400 can relay a desired imposed pressure difference to one ormore controllers 220, which then impose the time-dependent pressuredifference onto the fluid.

The methods disclosed herein account for reactions occurring within thedevice when calculating the value of the condition within therecirculating microfluidic device. The reaction can be a reaction of acomponent within the fluid, either via surface reactions, volumereactions, or bulk reactions. Surface or volume reactions include anyreaction taking place at any two or three-dimensional surface orconstruct within the microfluidic device. In some implementations of themethod, surface or volume reactions are assigned rate constants forcalculation purposes. The reaction can take place directly between acomponent of the fluid and the surface (i.e. adsorption/absorption), orthe reaction can take place between a component of the fluid and asurface component 232 that is attached to any surface within themicrofluidic device (for example, the binding of a component of thefluid to the antibodies/surface component 232 of FIG. 3 ). Surface orvolume reactions can be based on, or affected by, the surface or volumedensity of the surface component 232, the surface area, the spatiallocation of the surface within the microfluidic device, theconcentration of the component at the specific spatial location of thesurface, a concentration profile over a specified spatial region aroundthe surface, an effective flow rate of the fluid between the first andsecond reservoirs, time dependent mass transport rates, time dependentshear rates, time dependent shear stresses, time dependent flow rates offluid among the microfluidic chambers, and the average molecularvelocity of the component in the fluid. Surface or volume reactions canresult in the generation or depletion of a biological molecule. Thebiological molecule can be, for example, a product of an immobilizedenzyme. The biological molecule can also be, for example, cytokines ormetabolites produced by a cell culture on a surface within themicrofluidic device. For example, in the microfluidic device 200 shownin FIG. 2 , the liver cell culture in three-dimensional construct 224can generate metabolites that then are depleted by the motoneurons onthe microelectrode array 228 and/or the muscle cells on the cantileverarray 230. The metabolites could also be adsorbed to the surfaces of thechambers 206, the fluid path 208, or the first and second reservoirs202, 204. All of these surface or volume reactions are accounted for inthe method of calculating the value of a condition within therecirculating microfluidic device.

Bulk reactions include reactions that take place within the fluid. Forexample, bulk reactions can occur between a first component of the fluidand any other component of the fluid. The reactions can be reversible orirreversible. The bulk reactions can be based on, or affected by, one ormore of the following variables: the concentration of the component atthe specific spatial location within the fluid, a concentration profileover a specified spatial region within the fluid, an effective flow rateof the fluid between the first and second reservoirs, time dependentmass transport rates, time dependent shear rates, time dependent shearstresses, time dependent flow rates of fluid among the microfluidicchambers, and the average molecular velocity of the component.

Some implementations of the methods include accounting for convection ordiffusion of the component. Convection or diffusion can be affected byone or more of the following variables: the spatial location within thefluid, the concentration of the component at the specific spatiallocation within the microfluidic device, a concentration profile over aspecified spatial region within the fluid, an effective flow rate of thefluid between the first and second reservoirs, time dependent masstransport rates, time dependent shear rates, time dependent shearstresses, time dependent flow rates of fluid among the microfluidicchambers, the average molecular velocity of the component, and bulk orsurface or volume reactions taking place within the microfluidic device.This can include determining a variable diffusion-like term. Thediffusion-like term can be determined, for example, for at least onespecific spatial location within the microfluidic device. In someimplementations, the flow rate/fluid velocity for at least one specificspatial location is accounted for when determining the diffusion-liketerm. When the imposed pressure difference changes over at least onetime step, convection or diffusion can be accounted for before (orbefore and after) the at least one time step.

In some implementations, the method of calculating the value of acondition includes using a finite element method with boundaryconditions. In some implementations, the boundary condition is a valueof a condition that was determined by the method in a previous timestep. The boundary conditions are determined using analyticalcalculations, or determined via calculations from the previous time stepof the finite element method. The boundary conditions can be set, forexample, for an opening between at least one of the first or secondreservoir. The boundary conditions can include, for example, the imposedpressure difference, the effective pressure difference, the effectiveflow rate within the device, the first reservoir fluid volume, and/orsecond reservoir fluid volume. For example, the boundary conditions candepend, at least in part, on a hydrodynamic pressure difference (e.g.the difference in height between the two reservoirs, either due todifferent volumes, different heights on a tilting platform, or acombination thereof). In some implementations, the boundary conditionsinclude the first concentration (of the component in the firstreservoir), the second concentration (of the component in the secondreservoir), or both. The first concentration, the second concentration,or both can be determined, for example, using a volume weighted averageof i) the concentration of the component in the volume of fluid flowinginto or out of the first or second reservoir, and ii) the previous firstconcentration, the previous second concentration, or both. In someimplementations, a diffusion-like term is incorporated into the finiteelement method.

In some implementations, the value of the condition is determined usinganalytical methods. In some implementations, the condition is modeledusing a first order differential equation. The first order differentialequation is directly solved in the time domain, or an analyticalsolution can be determined in the frequency domain after performing aFourier transform. The variable diffusion-like term, described above,can be modeled within the analytical methods using ordinary differentialequations. In some implementations of the analytical methods, thechamber is modeled as a fluid resistor and the first and secondreservoirs are modeled as capacitors. The modeling of the first andsecond reservoirs can include incorporating a first order differentialequation into the analytical methods. In some implementations, thecalculations can be performed in a simulation software program, forexample, a software program that employs principles of electricalcircuit analysis.

Methods of performing an experiment using the recirculating microfluidicdevices are also disclosed herein. A flow chart schematic of anexemplary method 600 is shown in FIG. 6 . The methods include step 602of setting a desired value for a condition within the microfluidicdevice, step 604 of performing a calculation that accounts for aneffective pressure difference between the first reservoir and the secondreservoir and reactions occurring within the microfluidic device, andstep 606 of determining a set of initial conditions for achieving thedesired value. The set of initial conditions can include one or more ofa first reservoir fluid volume, a second reservoir fluid volume, a firstconcentration of a component in the first reservoir, a secondconcentration of the component in the second reservoir, and a timedependent imposed pressure difference. The initial conditions are thenused to initiate the experiment in step 608. Initiating the experimentcan include mixing the component into a solution to create a first fluidhaving the determined first concentration of the component. Initiatingthe experiment can further include adding a first volume of the firstfluid to the first reservoir (the first volume of the first fluid beingequivalent to the determined first reservoir fluid volume). In someimplementations, initiating the experiment can include mixing thecomponent into a solution to create a second fluid having the determinedsecond concentration of the component and adding a second volume of thesecond fluid to the second reservoir (the second volume of the secondfluid being equivalent to the determined second reservoir fluid volume).Finally, initiating the experiment can include imposing the determinedtime dependent imposed pressure difference to initiate circulation andrecirculation of the fluid. In some implementations, performing theexperiment further comprises sending instructions from a processor to acontroller to cause the controller to impose the determined timedependent imposed pressure difference on the fluid.

Although the subject matter has been described in language specific tostructural features and/or methodological acts, it is to be understoodthat the subject matter defined in the appended claims is notnecessarily limited to the specific features or acts described above.Rather, the specific features and acts described above are disclosed asexample forms of implementing the claims.

Example

Models and algorithms for fluid and mass transport with bulk and surfacereactions in recirculating microfluidic systems are described below. Themicrofluidic systems utilize recirculation of fluid via, for example, anoscillatory pressure differential between two or more fluidicreservoirs. This device is a microfluidic device containing fluid pathsand chambers of larger volumes with the chambers enabling theincorporation of various functional units. A functional unit can be aconstruct that is either responsive to environmental conditions,influences environmental conditions, or affects the fluidics within thesystem. For instance, a functional unit can include enzymes, catalysts,immobilized reactants or sensors. This system has applications forbody-on-a-chip (or organ-on-a-chip) devices, in which the functionalunits inside the chambers are cultures of cells and/or bioMEMS devicesor environmental sensors (e.g. dissolved gasses, soluble compounds,ions, pH, etc.). In some implementations, the fluid of the system aserum-free medium.

The oscillatory pressure differential used to drive the fluidic flow maybe imposed by a tilting platform to produce gravitational pressuredifferences, gas pressure to produce hydraulic pressure differences, orany such system that produces a time dependent oscillatory pressuredifferential. The imposed gravitational or hydraulic pressure may haveany regular profile (eg sawtooth, sinusoidal, square wave, etc) or evena random or non-regular profile that varies in time. This profile neednot return to the original position, such that the recirculation offluid may not be a complete recirculation. Additionally, this method andalgorithm may be used for modeling fluid and mass transport for aportion of an oscillatory cycle, a profile with a single change ofpressure, or any other profile that does not oscillate but which has animposed pressure that has at least one period of time in which thepressure is different from the initial condition.

The algorithm described herein incorporates finite volumes andconcentrations in two or more fluidic reservoirs as well as the timedependent imposed pressure differential. Because the reservoirs havefinite volumes of fluid and the microfluidic device has a fluidicresistance to flow, the effective pressure difference between the two ormore reservoirs is not linearly proportional to the imposed pressuredifferential. Instead, the effective pressure differential, whichcontrols the flow rate through the system, is dependent on thetime-dependent profile of imposed pressure differential and thetime-dependent hydraulic pressure difference between the reservoirscaused by the difference in volumetric height between the tworeservoirs. The mass transport through the system is related to the flowrates in the system as well as the concentrations of the components inthe reservoirs and throughout the microfluidic device. In particular,the finite reservoir volumes with finite concentrations of chemicalcomponents influences the mass transport through time dependent flow andthrough mixing and time-dependent concentrations within the reservoirsdue to fluid flowing into and out of these reservoirs. Additionally,reactions occurring in the system are modeled and both influence theoverall mass transport and are influenced by the microfluidic masstransport. These reactions may be surface reactions (e.g. immobilizedenzyme on a surface or monolayer of cells), reactions in certain volumes(e.g. immobilized enzyme in a 3D construct or material, or cellssuspended in a 3D construct or material such as a hydrogel or otherbiomaterial scaffold), or bulk reactions (e.g. conjugation of two ormore compounds in solution, or reactions between two or more solublecomponents). Adsorption and absorption of compounds to the microfluidicdevice are modeled, and are also influenced by and influence the masstransport throughout the system. Additionally, these reactions andadsorption/absorption may occur within the microfluidic device(including the fluidic channels and chambers) or within the reservoirs.For analytical purposes, the fluidic channels and chambers (which do notvary in volume of fluid over time) can behave as resistive elements tofluidic flow, while the reservoirs, which have a time-dependent volumeof fluid, can have a first order differential equation component to thefluidic response, and can act similar to a capacitor.

The algorithm can be enacted in in at least two ways: 1) computationallycalculated during each time step of a computational fluid dynamics orfinite element method analysis of the system or 2) calculatedanalytically using equations analogous to resistive and capacitiveelements.

In an example implementation of the computational calculation, in eachiteration, the imposed driving pressure difference P(t) (e.g.hydrodynamic pressure from tilting the device causing a difference inheight between two or more reservoirs) is altered by the hydrodynamicpressure difference between the two or more reservoirs caused by thedifference in volumes in the reservoirs. This altered pressuredifference is then imposed as boundary conditions on the computationalmodel at locations where liquid flows into and out of the reservoirsinto the microfluidic device. Additionally, the concentrations ofchemical components in the reservoirs are imposed on the same boundariesto define the concentrations of these components in the fluid flowinginto the microfluidic device. The volume for each reservoir for the nexttime step in the computational analysis is calculated from thevolumetric flow during the time step through the boundary between thereservoir and the microfluidic device. The concentration of thecomponents in the fluid flowing through these same boundaries is usedalong with the change in volume to determine the new concentrations ofcomponents within the reservoirs by performing a volume-weighted averageof the previous concentrations in the reservoir with the concentrationsin the volume of fluid flowing into or out of the reservoir.

In an example implementation of the analytical calculation, theeffective pressure differences between reservoirs and effective flowrates are modeled using first order differential equations eitherdirectly solving the differential equation in the time domain,determining the analytical solution in the frequency domain after aFourier transform, or performing these calculations in a simulationsoftware that incorporates these calculations (e.g. an electricalcircuit analysis software). Either the effective pressure differences orthe effective flow rates calculated from this method may be imposed onthe computational model as boundary conditions to produce equivalentsolutions.

For reactions at specific locations (for example, for metabolism ofcompounds by a liver or other metabolically active construct or thegeneration of cytokines or other signaling molecules) surface and volumereactions can be imposed on specific surfaces or on volumes representinga three dimensional construct. These reactions have specific rateconstants that may occur in a number of forms including zero order,first order, and second order equations, and these reactions may bereversible or irreversible. Adsorption and absorption of compounds ontosurfaces and into volumes is also modeled with reversible orirreversible reaction kinetics. Reactions within the bulk of the fluidare also modeled with reversible or irreversible reaction kinetics. Thetime-dependent flow dynamics and local concentration profiles caused bythe reservoir-based microfluidic device influence each of thesereactions and dynamics.

In one implementation of the invention, the transport of componentswithin the fluid due to the oscillatory imposed pressure difference ismodeled after each oscillation cycle. In this algorithm, the transportwithin the device during each cycle due to convection and diffusion ismodeled using a variable diffusion-like term. This diffusion-like termvaries within the model depending on the fluid velocity at a specificphysical location over the entire cycle. The velocity profilesthroughout the microfluidic device, coupled with the diffusivity of thecomponent within the fluid, are modeled or determined experimentally andthen the diffusion-like term as a function of position is determined foreach point within the microfluidic device. This diffusion-like term isthen used to calculate the transport of components within the fluid overtime using ordinary differential equations, and can be calculatedanalytically or computationally (e.g. finite element analysis). In thisimplementation, reaction rates and adsorption/absorption reactions areincorporated as above, to produce a model that is capable of modelingthe fluid dynamics and mass transport throughout the microfluidicdevice.

1-98. (canceled)
 99. A method of performing an experiment using amicrofluidic device with recirculation, the microfluidic devicecomprising a fluid, a first reservoir, a second reservoir in fluidcommunication with the first reservoir, at least one chamber in fluidcommunication with the first and second reservoirs, and a fluid pathfluidically connecting the first reservoir, the chamber, and the secondreservoir, the method comprising: setting a desired value for acondition within the microfluidic device, performing a calculation thataccounts for an effective pressure difference between the firstreservoir and the second reservoir and reactions occurring within themicrofluidic device, determining a set of initial conditions to achievethe desired value, wherein the set of initial conditions comprises oneor more of a first reservoir fluid volume, a second reservoir fluidvolume, a first concentration of a component in the first reservoir, asecond concentration of the component in the second reservoir, and atime dependent imposed pressure difference, and initiating theexperiment using the set of initial conditions.
 100. The method of claim99, wherein initiating the experiment comprises mixing the componentinto a solution to create a first fluid having the determined firstconcentration of the component.
 101. The method of claim 99, whereininitiating the experiment further comprises adding a first volume of thefirst fluid to the first reservoir, the first volume of the first fluidbeing equivalent to the determined first reservoir fluid volume. 102.The method of claim 99, wherein initiating the experiment comprisesmixing the component into a solution to create a second fluid having thedetermined second concentration of the component.
 103. The method ofclaim 99, wherein initiating the experiment further comprises adding asecond volume of the second fluid to the second reservoir, the secondvolume of the second fluid being equivalent to the determined secondreservoir fluid volume.
 104. The method of claim 99, wherein initiatingthe experiment further comprises further comprising imposing thedetermined time dependent imposed pressure difference on the fluid toinitiate circulation and recirculation of the fluid.
 105. The method ofclaim 99, wherein performing the experiment further comprises sendinginstructions from a processor to a controller to cause the controller toimpose the determined time dependent imposed pressure difference on thefluid.